Abstract: For decades, classic genetic model organisms have provided an unbiased and comprehensive means to functionally annotate the genome-to connect genotype with phenotype. Despite immense efforts, somatic cell genetic screens in human model systems have remained unattainable. Recently, however, the discovery of RNA interference-based functional screening provided an analogous discovery platform for human cells, one that continues to revolutionize our understanding of genome function, both as it relates to normal cellular biology and human disease. That said and while still appreciating it as an invaluable asset, RNA interferencebased screens are expensive and fraught with artifact, and therefore will never reach the comprehensive functional annotation achieved in yeast, Drosophila, C-elegans and zebrafish. The principle goal of this proposal is to realize a somatic cell genetic screening platform in human cells. We will accomplish this goal through the establishment and employment of stable near-haploid human cells, which exist in our laboratory. Retroviral insertional mutagenesis of these cells provides a means to quickly and inexpensively identify functional units within the human genome that individually and concertedly control phenotype. The clonal derivation and archiving of mutagenized near-haploid cells will yield a library of gene-compromised human cell lines, analogous to that of the yeast deletion libraries. I will leverage my existing expertise in proteomic and functional genomic analysis of signal transduction to validate the haploid screening approach. A systems-level integration of the resulting functional data with protein-protein interaction networks, genome-wide association data and transcriptional signatures will reveal targets of diagnostic, prognostic and therapeutic value. Success promises rapid, unbiased and inexpensive complete loss-of-function phenotypic screening of human cells, and therefore will transform the experimental strategies taken in both basic and applied sciences. Public Health Relevance: A significant challenge in medical science is identifying the full complement of genes that functionally contribute to specific cellular and disease processes. Although such genome annotation is easily achieved in classic genetic model systems, analogous approaches in human systems are expensive and fraught with false discoveries. We have designed an inexpensive and artifact-free functional genomic discovery platform specifically for mammalian cells, one that ascribes function to every human gene in the context of specific cellular behaviors, and more importantly one which promises to enhance the translational potential of our discoveries.